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MEDCHEM 562P 2014 - VITAMINS and Minerals
Part 1: Water Soluble Vitamins
Bill Atkins Ph.D. H172Q, 685 0379; [email protected] INTRODUCTION I. Who takes vitamin/mineral supplements and why? [Bailey et al. JAMA Intern
Med 173:355 (2013].
• It is estimated that ~50% of the adult population in the US takes some type of dietary supplement, typically to ‘improve/maintain overall health’.
• >75% of products are used without care-provider recommendation.
2
II. Multivitamins
It is estimated that ~30% of the adult US population take multivitamins daily. A recent clinical trial of male physicians taking multivitamins concluded there was a
very modest, but (statistically) significant, reduction in total cancers with daily multivitamin use (NEJM, 308:1871 (2012).
Do we need to supplement diets with vitamins/multivitamins? Maybe…. in certain circumstances.
III. When are vitamin supplements worthwhile?
Inadequate intake -- alcoholics, poor, elderly, dieters, poor diet
Increased needs -- pregnancy, lactation, infants, smokers, injury, trauma, recovery from surgery, infection
Poor absorption -- elderly, GI disorders, specific GI surgeries, e.g. gallbladder
removal, gastric bypass, cystic fibrosis, severe diarrhea, drug-induced vitamin deficiencies – e.g. long term antibiotic use, cholestyramine, mineral oil
IV. Definitions
• Vitamins are organic compounds and minerals are chemical elements that are required as nutrients in small amounts by an organism.
• Vitamers are different forms of a particular vitamin, e.g. vitamins K1 and K2, vitamins D2 and D3, retinol and retinal, etc.
3
V. Reference Tables for Intake of Vitamins and Elements Intakes DRI Reports are produced by the Food & Nutrition Board of the Institute of
Medicine, National Academies of Science. http://fnic.nal.usda.gov/dietary-guidance/dietary-reference-intakes/dri-tables
Estimated average requirements (EAR): the average daily nutrient intake level estimated to meet the requirements of half of the healthy individuals in a group.
Recommended Dietary Allowance (RDA): the average daily dietary intake level; sufficient to meet the nutrient requirements of nearly all (97-98%) healthy individuals in a group. Calculated from the EAR.
Tolerable Upper Limit (UL): maximum adult daily intake unlikely to cause harm.
RDA= 1.2(EAR)
Daily Values (DVs) are set by the FDA.
http://ods.od.nih.gov/HealthInformation/dailyvalues.aspx • Two groups: Daily Reference Values (DRVs) for energy-producing nutrients,
e/g. fats, carbohydrates, protein etc. and Reference Daily Intakes (RDIs) for vitamins and minerals.
• A DV is often, but not always, similar to one's RDA for that nutrient. • DV is primarily used for labeling purposes. % DV on label is based on 2,000
calorie/day diet for adults and children over 4 yrs.
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VI. Standardization
• Units of biological activity (IU) superceded, where known, by potencies based on weight (µg, mg) of the most active vitamer.
• IoM guidelines use weight. • FDA labels use both.
VII. History and Discovery 1500 BC Ancient Egyptians used liver - rich in vitamin A - applied to the eye to treat night
blindness.
1536 Jacques Cartier, exploring the St. Lawrence River, uses local native knowledge to save his men from scurvy by boiling the needles from cedar trees to make a vitamin C-rich tea.
1795 British navy adds lemons to sailors' rations, 40 years after a Scottish naval surgeon, James Lind, had urged that citrus fruits be used to prevent scurvy.
1884 Japanese navy eradicates beriberi - vitamin B1 deficiency- by feeding sailors meat and fruit in addition to polished white rice, which lacked the thiamine-rich husks.
1897 Beriberi (a polyneuritis) was induced in birds fed only polished rice and reversed by feeding the rice polishings.
1911 Casimir Funk isolates amine-containing concentrate containing thiamine from rice polishings, which was curative for polyneuritis in pigeons, and names it 'vitamine' for vital amine.
5
1912 Xavier Mertz –Antarctic explorer – dies of vitamin A poisoning from ingesting sled dog liver after supplies are lost in a crevasse.
Year discovered Vitamin Source
1912 Vitamin B1 Rice bran
1912 Vitamin C Lemons
1913 Vitamin A Milk/egg yolk
1918 Vitamin D Cod liver oil
1920 Vitamin B2 Eggs
1922 Vitamin E Wheat germ, Seed oils
1926 Vitamin B12 Liver
1929 Vitamin K Alfalfa
1931 Vitamin B5 Liver
1931 Vitamin B7 Liver
1934 Vitamin B6 Rice bran
1936 Vitamin B3 Liver
1941 Vitamin B9 Liver
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VII. WATER SOLUBLE VITAMINS
A. Generalities 1. Metabolism and storage -- only B12 and folate are appreciably stored. In
general, water soluble vitamins are excreted readily and are not stored. As a result, depletion is more of a problem than toxicity.
2. Toxicity -- only niacin and pyridoxine are at all toxic (in high conc.). In general, the water soluble vitamins have few toxicities. The water soluble vitamins are coenzymes for various common biochemical reactions and their status can be readily determined by measuring the appropriate enzyme activities in blood. Typically, the enzyme activity is measured in the absence and the presence of exogenously added coenzyme, to determine whether the patient needs more of the vitamin.
B. Thiamin -- (Vitamin B1) 1. Structure
TMPN SCH2
CH3 N
N
CH3 CH2 CH2OH
Hthiamin
ATP ATPTPP
Chemically, the structure includes a pyrimidine ring and a thiazole ring. The thiazole ring has received enormous attention from mechanistic biochemists - they have pondered the reason for the choice by nature of a sulfur atom in the ring. Imidazole rings and oxazole rings would also, in principle, be capable of catalyzing the relevant chemical reactions.
Space-filling model of Thiamin
7
2. Function – used to harvest carbohydrates for energy a) Oxidative decarboxylation of α-keto acids
+
CH3 C CO2HO
TPP
lipoic acidNAD
CoASHpyruvate dehydrogenase complex
CH3 C SCoAO
CO2e.g. + + NADH
e.g. α -ketoglutaric acid TPP succinyl CoA CO2
NADCoASH
++
lipoic acidα-ketoglutarate dehydrogenase complex
8
The decarboxylation is accomplished by a mitochondrial enzyme complex as shown below. L = lipoic acid, E = enzyme, TPP = thiamin pyrophosphate. Pyruvate dehydrogenase complex in detail
b) Transfer of α-ketols (pentose phosphate pathway) -- 10% of carbohydrate metabolized this way. This pathway provides pentoses for RNA and DNA synthesis and NADPH for the biosynthesis of fatty acids and other endogenous reactions.
c) Non-coenzyme function – TTP involved in the control of chloride channels in brain and elsewhere in nerve impulse conduction.
E LSH
SH
CoASH
CH3 CSCoAO
NAD+
TCAcycle
E TPP
E TPP
C OH
CH3
(α-hydroxyethyl TPP)
E LS
S
E L SH
S C CH3O
CH3 C COOHO
CO2
NADH
CHOHCHO
CH2OPO3H2
+O=
HOCHCCH2OH
HCOH
HCOH
HCOHCH2OPO3H2
CHOHCHOH
CHOH
CHO
CH2OPO3H2
+O=
HO CHCCH2OH
HC OHCH2OPO3H2
TPPtransketolase
e.g.
xyulose-5-phosphate ribose-5-phosphate
sedoheptulose-7-phosphate
glyceraldehyde phosphate
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3. Mechanism – formation of adduct with C2 of thiazole ring. ‘Classic’ experiments support this mechanism, including facile exchange of solvent D2O at the C-2 position.
4. Deficiency – thiamin needs are proportional to caloric intake and is essential for carbohydrate metabolism. Usually consider requirement as 0.5 mg/1000 calories plus 0.3 mg during pregnancy and lactation. Studies show laboratory evidence of thiamin deficiency (erythrocyte transketolase assay) in 20-30% of elderly patients and 40-50% of chronic alcoholics. <2% of healthy controls showed evidence of deficiency. a) Early signs of deficiency – anorexia, nausea, vomiting, fatigue,
weight loss, nystagmus, tachycardia.
b) Late signs of deficiency – Beriberi cardiac - increased heart size, edema cerebral - depression, irritability, memory loss, lethargy GI tract - vomiting, nausea, weight loss neurological - weakness, polyneuritis, convulsions. Signs and symptoms vary with age of patient, rapidity of onset, and severity of deficiency.
+H
+H
N S
CCH3
OH
NC
S
CHCH3
OH
NC
S
H
H+ + CH3 CHO
α-hydroxy-ethyl-TPP
H+
N S
CCH3 OH
C OO
3 2 H++NC
S
H
NC
S
CH3 C COOH
O
NC
Spyruvate
H+
CH3 C COOH
O
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c) Thiamine and the alcoholic (i) intake low and alcohol blocks conversion of thiamin
→ TPP (ii) ↓ absorption - ↓ active transport (iii) ↓ storage (iv) increased fluid intake and urine flow causes thiamin
washout (v) involved in fetal alcohol syndrome.
d) Wernicke-Korsakoff syndrome – seen in some alcoholics;
neurological disorder resulting in impaired mental functioning → institutionalization for a significant number of patients. Symptoms: confusion, memory loss, confabulation, psychotic behavior; maybe irriversible in part.
e) Factors → B1 deficiency (i) ↑ carbohydrate intake -- TPN, alcoholics (ii) ↓ absorption -- ulcerative colitis, etc., alcoholism (iii) ↓ intake -- poor diet, geriatrics, breast fed infant from B1
deficient mother, etc. (iv) alcoholism.
f) Cellular uptake – Intestinal cells contain a thiamin specific
receptor/transporter (hTHTR) which appears to specifically pump thiamin and not TPP. After cellular uptake, thiamin is converted to TPP. Polymorphisms in the gene encoding hTHTR are known and may contribute to thiamin-responsive megaloblastic anemia. Once in the circulation specific transport proteins may ‘store’ TPP and control its circulation (hypothesized), but most is bound to Albumin. Most ends up in skeletal muscle, brain, heart, liver.
5. Source – present in most tissues (as TPP) and plants (as thiamin); rich sources include: lean meat, especially pork, cereal grains, eggs, yeast, nuts. Thiaminase – in some fish (raw) and shellfish (raw) and ferns. This enzyme can hydrolyze thiamin.
NC
SCH2R
In the milling or processing or processing of rice and flour, the thiamin is lost. ‘Whole’ wheat or rice contains ~10 times as much Thiamin as white wheat or rice. Today in the USA, most white flour, rice and pastas are "enriched" to bring thiamin levels to near original levels. "Enriched" products also have added riboflavin, niacin, iron, and folic acid.
11
6. Stability – labile at pH > 4 and when heated (especially at alkaline pH values) prolonged cooking ¯ levels especially at pH > 4.
7. Diagnosis of deficiency state a) ↑ pyruvate and lactate in plasma b) transkelotase activity in RBC – most important technique.
8. Uses
a) deficiency states -- for alcoholics b) thiamin responsive inborn errors of metabolism -- see below c) mosquito repellant -- efficacy? -- for dogs, for humans 50 mg QID
2d before and during exposure is recommended. d) acute alcoholism: give 100 mg IM or IV stat. This is a common
practice. e) Alzheimer’s disease—little evidence for benefit (huge doses used).
9. Thiamin responsive inborn errors of metabolism:
Disease Defect Wernicke – Korsakoff Transketolase Maple Syrup urine disease Failure to decarboxylate branched chain amino acids Thiamin responsive megaloblastic anemia Hyperalanemia ? Hyperpyruvate acidurea Pyruvate dehydrogenase
10. Requirement – 0.5 mg/1000 cal. DV = 1.5 mg. Minimum intake should be at least 1 mg.
11. Toxicity – nontoxic on oral administration; No UL value. Anaphylactic reactions have been observed in patients receiving repetitive parenteral doses.
12. Patient Counseling/ Patient Use Issues a) Needed to drive carbohydrates to energy b) Rarely needed as a single supplement. Use a multivitamin to get
needed thiamin. c) Special benefit in alcoholics at higher doses d) Benefit in high doses in rare thiamin-responsive inborn errors of
metabolism e) Uncertain benefit as a mosquito repellant (if true, 1-2 week onset) f) Nontoxic.
12
C. Riboflavin (Vitamin B2) Like Vitamin B1, Riboflavin is highly water soluble, and it is difficult to achieve toxic levels in the body – excess vitamin is efficiently eliminated renally. It used as an additive in many enriched foods and can be produced in huge, industrial scale, quantities by expression of the biodynthetic enzymes in fungi or bacteria. These genetically engineered organisms may appear bright yellowish-orange, which is the color of Riboflavin. 1. Structure
riboflavin coenzymesFAD = flavin adenine dianucleotideFMN = riboflavin monophosphate
N
NNH
N
O
OCH3
CH3
CH2 CH CH CH CH2OHOH OH OH
gut mucosa liverriboflavin FMN FADATP ADP ATP ADP
13
2. Function – redox, tissue respiration, H transfer as flavin containing enzyme. oxidized – yellow reduced - colorless Examples of enzymes having flavin groups: succinate dehydrogenase (-succinate → fumerate in TCA cycle) fatty acid acyl CoA dehydrogenase (β-oxidation of lipids) glutathione reductase – important in antioxidant activities The following are important flavoproteins (containing FMN): Cytochrome C reductase (electron transport); NADP+ -- cytochrome C reductase; cytochrome P-450 reductase (drug metabolism), flavin monooxygenase (drug metabolism).
3. Deficiency state a) Not usually seen in isolation but occurs in combination with other
B vitamin deficiencies. b) Fatigue, cheilosis, glossitis, vascularization of cornea, dermatitis c) Vegans and teenagers may be low in B2 if dairy intake is low d) Low B2 intake may be a risk factor for cataract development e) Alcoholics are at risk due to low intake and low absorption.
4. Source – milk, meats, leafy vegetables, eggs, yeast, “enriched” products.
5. Stability
a) Usually > 30% destroyed by cooking b) Labile to light
red, colorlessox-yellow
R
NH
NH
HN
RO
O
RN
NNH
N O
O-2H⋅
⋅+2H
NADPH
NADP
FAD
FADH2
2GSH
GSSG
H2O2 or ROOH
2H2O or ROH + H2O
glutatione peroxidaseglutathionereductase
14
c) More stable in acid than alkali in absence of light.
6. Use a) Deficiency states. Is a component of most multivitamin mixtures. b) New – may help in migraine headache prevention. c) New – high intake associated with lower risk for cataracts and a
3mg supplement reduced risk.
7. Requirements a) DV = 1.7 mg b) “Average” U.S. diet contains 2 mg for males and 1.5 mg for
females c) Diagnosis – erythrocyte glutathione rreductase activity d) No UL value
8. Patient Counseling/ Patient Use Issues
a) Routine single dose supplementation not needed. Use a
multivitamin to get needed riboflavin. b) Possible use in preventing migraine headaches. Use 400 mg/d. c) Will turn urine bright yellow in doses higher than the DV. d) Nontoxic.
D. Vitamin B6 1. Structure
Pyridoxine is a commonly used term for this vitamin, but all 3 are equally active so vitamin B6 is a better term to use. Three phosphorylated forms are present also: P PO4 PLP
FMNPNP
FMN
pyridoxamine (PN)
N
CH2OHHO
CH3
CH2NH2
N
CH2OHCH2OHHO
CH3
pyridoxine (P)
N
CH2OHHO
CH3
CHO
pyridoxal (PL)
15
Coenzyme = pyridoxal-5-phosphate “PLP”
2. Function – participates in over 140 enzymatic reactions by forming a Schiff base with the terminal amino group of lysine in the enzyme. It is estimated that this corresponds to ~4% of all enzymatic reactions known. a) Transamination
e.g. glutamate-aspartate transaminase
N
CH2OP
CH3
HOCH
O
R NH2
holoenzymeNCH3
HO CH2OPCHNR
PLP
PLP dependent enzyme
+
R C COOHO
α-keto acid #1
N
HO CH2OP
CH3
CHNenzyme
N
HO CH2OP
CH3
CHNCHR COOH
N
HO CH2OP
CH3
CHNCR COOHH
N
HO CH2OP
CH3
CHNH2
R CH COOHNH2
amino acid #1
+
+
H2O
+ R´ C COOHNH2
amino acid #2
+ R´ C COOHO
α-keto acid #2N
CH2
NH2
HO
CH3
CH2OP
N
HO
CH3
CH2OPCHO
PLPPNP
PLP
oxaloacetic acid alanineCH3 CH COOH
NH2
transaminasepyruvic acid
HOOC CH2 C COOHO
+
aspartic acid HOOC CH2 CH COOH
NH2+ CH3 C COOH
O
16
b) Decarboxylation e.g. e.g. e.g. e.g.
Note: B6 contraindicated in levo-DOPA therapy because B6 enhances peripheral decarboxylation of levo-DOPA to dopamine which will not cross Blood Brain Barrier; Larobec® (Roche) contains no pyridoxine and can be used if multivitamin supplementation is desired for patient on l-DOPA. The anti-Parkinsons’s drug Sinemet® contains levo-DOPA and carbidopa (A DOPA
N
HO CH2OP
CH3
CHO
N
HO CH2OP
CH3
CHNCHR COOH
N
HO CH2OP
CH3
CHO
CH2RNH2+
decarboxylase
H2O
amino acid
R CH COOHNH2 + + CO2
HOOC CH2 CH2 C COOHH
NH2PLP
CO2
HOOC CH2 CH2 C HH
NH2
glutamic acid γ-amino butyric acid (GABA)
N
HOC COOHH
NH2
5-hydroxytryptophan
N
HOCH2
NH2
5-hydroxytryptamine (serotonin)
CO2
PLP
PLP
CO2NN
CH2HCH
NH2
histamine
NNCH2
HC COOHNH2
histidine
PLP
CO2 CH2 CH2
OH
NH2
HOHO
CH2HC COOHNH2
OH
DOPA DOPAMINE
17
decarboxylase inhibitor) -- therefore, no interaction.
c) B6 and sulfur amino acid metabolism. (Note: elevated homocysteine is an independent risk factor for cardiovascular disease and birth defects.)
d) B6 involvement in methionine formation (and S-adenosyl methionine) makes it indirectly involved in methylation. Hence B6 is indirectly involved in lipid metabolism and nucleic acid formation and immune function.
C COOHCH2
H
NH2
CH2SCH3
OCH2
S CH2NH2
HCH2 COOCH
adenine
OHOH
-
OCH2
CH3 S CH2 CH2 COOCH
adenine
OHOH
H
NH2
-
C COOHCH2
H
NH2
H3C
C COOHCH2
H
NH2
HS
α-ketobutyrate
cysteine
cystathionine
PLP serine
methionine
S-adenosylmethionine
methyl acceptor
S-adenosylhomocysteine
THFA
N5-methylTHFA
methyl B12
hydroxy B12
ATP
C COOHCH2
H
NH2
CH2HS
homocysteine
C COOHCH2
H
NH2
CH2S
CH2 C COOHH
NH2
+
PLPcystathionineγ-lyase
18
e) B6 involved in tryptophan metabolism to serotonin and niacin.
f) Other reactions requiring B6: (i) Glycogen phosphorylase (release of glucose in muscle) (ii) Heme biosynthesis
NiacinN
COOH
COOH
NH2NH
tryptophanoxygenase
serotonintryptophan
COOH
NH2NH
HO
N-formylkynurenine
NH2NCHO
COOH
O
3-hydroxykynurenine
NH2
COOH
OHNH2
O
kynureninasePLP
COOH
NH2OH
3-hydroxy anthranilic acid
NNH2
HO
Htryp
decarboxylase
PLP
19
(iii) Nucleic acid biosynthesis (via SAM)
SUMMARY of Pyridoxal/pyridoxamine reactions
CH2
N
C
N
HC
HC
N
N
B:
external imine internal imine
HO-
H2C
C
N
HN
OH
external carbinolamine
HC
HC
N
HN
HO-
OH
internal carbinolamine
H2C
N
NH2
OHC
N
O
NH2
hydrolysis of Imine leads to incorporation of ‘oxygen atom’ H2O attacks carbon atom of the imine, that carbon gets an oxygen PLP enzymes control which carbon atom gets the oxygen atom by switching between the external imine and the internal imine.
20
3. Deficiency
a) Not seen usually and, if seen, is associated with other vitamin
deficiencies or is iatrogenic; symptoms include rash, peripheral neuritis, anemia and possible seizures. Deficiency diagnosed by low plasma PLP and low transaminase activities (±PLP).
b) Iatrogenic B6 deficiencies (i) Isoniazid – antituberculosis drug – forms Schiff base with
B6. Can get neuritis and convulsions. 25-300 mg/d B6 given to prevent B6 deficiency.
(ii) 4-Deoxypyridoxine – (experimental only)
N
HOCH3
CH2OH
CH3 Symptoms – Skin lesions on face, glossitis, stomatitis, convulsive seizures (↓ GABA?), anemia (↓ heme synthesis?).
(iii) Oral contraceptives.- older high dose Ocs can affect B6 but not a problem now.
4. Source – milk, meats, legumes, tuna, whole grains, beans.
5. Stability – pyridoxine is stable; some loss on cooking, especially with meats, due to Schiff base formation and decrease of the pyridoxal in the foods.
N
CO
NH NH2
N
HCO
CH2OHHO
CH3
+
N
CO
NH N CHO CH2OH
H
CH3
NIsoniazid
21
6. Diagnosis of Deficiency – measure erythrocyte transaminases.
7. Use a) Routine use in multivitamin products b) In INH therapy c) Certain inborn errors of metabolism
Name Symptoms Dose of B6 Problem
B6--dependent infantile convulsions
Clonic and tonic seizures
10-25 mg/day
Defective glutamic acid decarboxylase; possible GABA depletion
B6--responsive anemia
Microcytic, hypochromic anemia
100 mg/day
Defective hemoglobin synthesis
Xanthurenic acidurea Mental retardation 25-100 mg/day Defective tryptophan metabolism due to faulty kyureninase, xanthurenic acid spills into urine
Homocystinurea Mental retardation Early heart disease
25-500 mg/day Defective cystathionine synthetase homocysteine appears in urine
Cystathionurea Mental retardation 25-500 mg/day Defective cystathionase d) PMS (50-500 mg/d) -- evidence is uneven. PLP is known to bind
to steroid receptors.
e) Carpal tunnel syndrome -- evidence is uneven. It seems to work for some. A trial of B6 100-200 mg/d for 6 mos. may be worthwhile.
f) Use in lowering homocysteine levels (see sulfur amino acid scheme above). High homocysteine may be an independent risk factor for cardiovascular disease but this is now controversial. Combine with folic acid and B12 for optimum lowering action.
g) Nausea and vomiting in pregnancy-Helpful in high doses. PremesisRx contains 75mg sustained release B6 (plus 12ug B12, 1mg folic acid and 200mg calcium) or 25mg of generic B6 TID is less expensive.
8. Requirement – DV = 2 mg; UL = 100 mg.
9. Toxicity a) > 200 mg/day can decrease prolactin levels b) > 1-2 g/day can cause serious neuropathy by an unknown
mechanism. Recommendation: avoid long term use in doses above
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200 mg.
10. Patient Counseling/ Patient Use Considerations a) Routine single dose supplementation usually not needed. Use a
multivitamin to get needed B6. b) Sometimes used, with limited evidence, for carpal tunnel
syndrome, PMS, and depression on OCs. c) Rare use in high doses for inborn errors. d) Sometimes used to prevent neuropathy with isoniazid. e) For nausea and vomiting of pregnancy, 25 mg TID or use
PremensisRx which is FDA approved for this. f) Used with B12 and folic acid in high homocysteine. g) Keep doses < 200 mg/d to avoid neuritis.